3&#39;-oh unblocked nucleotides and nucleosides base mdified with non-cleavable, terminating groups and methods for their use in dna sequencing

ABSTRACT

Provided are novel nucleotides, nucleoside, and their derivatives described herein, that can be used in DNA sequencing technology and other types of DNA analysis. In one embodiment, the nucleotide or nucleoside with an unprotected 3′-OH group is derivatized at the nucleobase to include a fluorescent dye attached via a linker to a non-cleavable terminating group. The non-cleavable-fluorescent group is designed to terminate DNA synthesis so that DNA oligomers can be sequenced efficiently in a parallel format. These reagents and methods will lead to more accurate identification of polymorphisms and other valuable genetic information.

FIELD OF INVENTION

The present invention relates generally to compounds and methods for DNAsequencing and other types of DNA analysis. More particularly, theinvention relates to nucleotides and nucleosides labeled withnoncleavable groups and methods for their use in DNA sequencing andanalysis.

BACKGROUND

Methods for rapidly sequencing DNA have become needed for analyzingdiseases and mutations in the population and developing therapies. Themost commonly observed form of human sequence variation is singlenucleotide polymorphisms (SNPs), which occur in approximately 1-in-300to 1-in-1000 base pairs of genomic sequence. Building upon the completesequence of the human genome, efforts are underway to identify theunderlying genetic link to common diseases by SNP mapping or directassociation. Technology developments focused on rapid, high-throughput,and low cost DNA sequencing would facilitate the understanding and useof genetic information, such as SNPs, in applied medicine.

In general, 10%-to-15% of SNPs will affect protein function by alteringspecific amino acid residues, will affect the proper processing of genesby changing splicing mechanisms, or will affect the normal level ofexpression of the gene or protein by varying regulatory mechanisms. Itis envisioned that the identification of informative SNPs will lead tomore accurate diagnosis of inherited disease, better prognosis of risksusceptibilities, or identity of sporadic mutations in tissue. Oneapplication of an individual's SNP profile would be to significantlydelay the onset or progression of disease with prophylactic drugtherapies. Moreover, an SNP profile of drug metabolizing genes could beused to prescribe a specific drug regimen to provide safer and moreefficacious results. To accomplish these ambitious goals, genomesequencing will move into the resequencing phase with the potential ofpartial sequencing of a large majority of the population, which wouldinvolve sequencing specific regions or single base pairs in parallel,which are distributed throughout the human genome to obtain the SNPprofile for a given complex disease.

Sequence variations underlying most common diseases are likely toinvolve multiple SNPs, which are dispersed throughout associated genesand exist in low frequency. Thus, DNA sequencing technologies thatemploy strategies for de novo sequencing are more likely to detectand/or discover these rare, widely dispersed variants than technologiestargeting only known SNPs.

Traditionally, DNA sequencing has been accomplished by the “Sanger” or“dideoxy” method, which involves the chain termination of DNA synthesisby the incorporation of 2′,3′-dideoxynucleotides (ddNTPs) using DNApolymerase (Sanger, F., Nicklen, S., and Coulson, A. R. (1977) DNAsequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA74, 5463-5467). The reaction also includes the natural2′-deoxynucleotides (dNTPs), which extend the DNA chain by DNAsynthesis. Balanced appropriately, competition between chain extensionand chain termination results in the generation of a set of nested DNAfragments, which are uniformly distributed over thousands of bases anddiffer in size as base pair increments. Electrophoresis is used toresolve the nested DNA fragments by their respective size. The ratio ofdNTP/ddNTP in the sequencing reaction determines the frequency of chaintermination, and hence the distribution of lengths of terminated chains.The fragments are then detected via the prior attachment of fourdifferent fluorophores to the four bases of DNA (i.e., A, C, G, and T),which fluoresce their respective colors when irradiated with a suitablelaser source. Currently, Sanger sequencing has been the most widely usedmethod for discovery of SNPs by direct PCR sequencing (Gibbs, R. A.,Nguyen, P.-N., McBride, L. J., Koepf, S. M., and Caskey, C. T. (1989)Identification of mutations leading to the Lesch-Nyhan syndrome byautomated direct DNA sequencing of in vitro amplified cDNA. Proc. Natl.Acad. Sci. USA 86, 1919-1923) or genomic sequencing (Hunkapiller, T.,Kaiser, R. J., Koop, B. F., and Hood, L. (1991) Large-scale andautomated DNA sequencing Determination. Science 254, 59-67;International Human Genome Sequencing Consortium. Initial sequencing andanalysis of the human genome. (2001) Nature 409, 860-921).

The need for developing new sequencing technologies has never beengreater than today with applications spanning diverse research sectorsincluding comparative genomics and evolution, forensics, epidemiology,and applied medicine for diagnostics and therapeutics. Currentsequencing technologies are too expensive, labor intensive, and timeconsuming for broad application in human sequence variation studies.Genome center cost is calculated on the basis of dollars per 1,000. Q₂₀bases and can be generally divided into the categories ofinstrumentation, personnel, reagents and materials, and overheadexpenses. Currently, these centers are operating at less than one dollarper 1,000 Q₂₀ bases with at least 50% of the cost resulting from DNAsequencing instrumentation alone. Developments in novel detectionmethods, miniaturization in instrumentation, microfluidic separationtechnologies, and an increase in the number of assays per run will mostlikely have the biggest impact on reducing cost.

It is therefore an object of the invention to provide novel compoundsthat are useful in efficient sequencing of genomic information in highthroughput sequencing reactions.

It is another object of the invention to provide novel reagents andcombinations of reagents that can efficiently and affordably providegenomic information.

It is yet another object of the invention to provide libraries andarrays of reagents for diagnostic methods and for developing targetedtherapeutics for individuals.

SUMMARY

Provided are nucleoside compounds as well as phosphates and saltsthereof, that can be used in DNA sequencing technology. The compoundsare optionally in the form of ribonucleoside triphosphate (NTP) anddeoxyribonucleoside triphosphate (dNTP) compounds. The nucleotide andnucleoside compounds include a noncleavable group labeled with afluorescent dye. The nucleotide and nucleoside compounds are designed toterminate DNA synthesis, so that nucleic acid oligomers can be sequencedrapidly in a parallel format.

A variety of nucleotide and nucleoside compounds, containing thenucleobases adenine, cytosine, guanine, thymine, uracil, or naturallyoccurring derivatives thereof, are provided that can be derivatized toinclude a detectable label such as a dye.

In one embodiment the base of the nucleoside is covalently attached witha benzyl group, and the alpha carbon position of the benzyl group isoptionally substituted with one alkyl or aryl group as described herein.The benzyl group can be functionalized to enhance the terminationproperties. The termination properties of the optionally alpha carbonsubstituted benzyl group attached to the nucleobase occur even when the3′-OH group on the ribose sugar is unblocked. These 3′-OH unblockedterminators are well-tolerated by a number of commercially available DNApolymerases, representing a key advantage over 3′-O-blocked terminators.The linker group also can be derivatized to include a selectedfluorescent dye.

In particular, methods for DNA sequencing are provided usingcombinations of the four nucleoside triphosphate compounds, modifiedwith a non-cleavable terminating group, and derivatives described hereinand labeled with distinct fluorescent dyes, which could be used foridentifying the incorporated bases to reveal the underlying DNAsequence.

DETAILED DESCRIPTION

Provided are nucleotide and nucleoside compounds as well as salts,esters and phosphates thereof, that can be used in rapid DNA sequencingtechnology. The compounds are optionally in the form cf ribonucleosidetriphosphates (NTPs) and deoxyribonucleoside triphosphates (dNTP). Thenucleotide and nucleoside compounds in one embodiment includes anon-cleavable group labeled with a fluorescent dye. The nucleotide andnucleoside compounds are designed to terminate DNA synthesis, so thatthese monomers can be used for rapid sequencing in a parallel format.The presence of such groups labeled with fluorescent dyes on thenucleotide and nucleoside compounds can enhance the speed and accuracyof sequencing of large oligomers of DNA in parallel, to allow, forexample, rapid whole genome sequencing, and the identification ofpolymorphisms and other valuable genetic information.

A variety of nucleotide and nucleoside compounds, containing thenucleobases adenine, cytosine, guanine, thymine, uracil, or naturallyoccurring derivatives thereof, are provided that include non-cleavableterminating moieties and/or which can be derivatized to include adetectable label such as a dye.

In one embodiment, the nucleobases adenine, cytosine, guanine, thymine,uracil, or naturally occurring derivatives thereof, can be covalentlyattached to a dye via a non-cleavable terminating moiety. Thenon-cleavable terminating moiety can be derivatized to enhance itstermination of DNA synthesis thus increasing its usefulness in DNAsequencing.

I. Advantages of Compounds for Sequencing

Nucleotide and nucleoside compounds are provided which are useful in DNAsequencing technology. The efficiency of incorporation of compoundsaccording to the invention may range from about 70% to about 100% of theincorporation of the analogous native nucleoside. Preferably, theefficiency of incorporation will range from about 85% to about 100%.Further, termination of nucleic acid extension will range from about 90%to about 100% upon incorporation of compounds according to theinvention. Nucleotide and nucleoside compounds in one embodiment have atermination efficiency of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%,or 99.9%.

II. Compounds

A variety of nucleosides and compounds as well as their mono, di andtriphosphates are provided. The compounds are useful for sequencingtechnology. In one embodiment, the nucleoside compound includes afluorescent group that can be detected efficiently. The nucleosidecompounds can be converted into their respective triphosphates for DNApolymerase reactions. Compounds according to the invention may berepresented by the

following formula:wherein R₁ is H, monophosphate, diphosphate or triphosphate, R₂ is H orOH, base is cytosine, uracil, thymine, adenine, guanine, or a naturallyoccurring derivative thereof, the non-cleavable terminating moiety is agroup imparting polymerase termination properties to the compound,linker is a bifunctional group, and the dye is a fluorophore. Compoundsaccording to the invention can be designed as fluorescent, non-labilenonreversible terminators useful in DNA synthesis sequencing.

In one embodiment, a compound is provided having a structure of formulasI-VII:

wherein R₁═H, monophosphate, diphosphate or triphophosphate, R₂═H or OH,R₃ and R₄ are each independently selected from the group of H, a C₁-C₁₂straight chain or branched alkyl, a C₂-C₁₂ straight chain or branchedalkenyl or polyenyl, a C₂-C₁₂ straight chain or branched alkynyl orpolyalkynyl, and an aromatic group such as a phenyl, naphthyl, orpyridine ring, R₅, R₆, and R₇, are each independently selected from thegroup H, OCH₃, NO₂, CN, a halide, a C₁-C₁₂ straight chain or branchedalkyl, a C₂-C₁₂ straight chain or branched alkenyl or polyenyl, a C₂-C₁₂straight chain or branched alkynyl or polyalkynyl, an aromatic groupsuch as a phenyl, naphthyl, or pyridine ring, and/or a linker group ofthe general structure:

X═CH₂, CH═CH, C≡C, O, S, or NH, Y═CH₂, O, or NH, n=an integer from 0-12;m=an integer from 0-12, and Dye=a fluorophore, and R₈ and R₉ are asdefined above for R₅, R₆, and R₇, with the proviso that R₈ and R₉ arenot NO₂, or pharmaceutically acceptable salt or ester thereof orenantiomer, racemic mixture, or stereoisomer thereof.

In a preferred embodiment, R₃ and R₄ are selected from the groupconsisting of, but not limited to, —CH₃, —CH₂CH₃, —CH₂CH₂CH₃, isopropyl,tert-butyl, and phenyl. Alternatively, R₃ and R₄ are selected from thegroup consisting of, but not limited to, alkyl and aromatic groupsoptionally containing at least one heteroatom in the alkyl or aromaticgroups, and further wherein the aromatic group may optionally be an arylsuch as phenyl or polycyclic such as a naphthyl group. In certainembodiments, R₅, R₆, R₇, and R₈ are selected from an aromatic groupconsisting of aryl and polycyclic groups.

Alternatively, linkers may have the following general structures:

For example, compounds with such linkers could have the followingstructures:

wherein the noncleavable terminating moiety can be attached to the basethrough a linkage such as a benzyl amine, benzyl ether, carbamate,carbonate, 2-(o-nitrophenyl)ethyl carbamate, and/or2-(o-nitrophenyl)ethyl carbonate.

Fluorescent dyes are not particularly limited. For example, thefluorophore may be selected from the group consisting of, but notlimited to, BODIPY, fluorescein, rhodamine, coumarin, xanthene, cyanine,pyrene, phthalocyanine, phycobiliprotein, alexa, squarene dye,combinations resulting in energy transfer dyes, and derivatives thereof.

Preferred embodiments include but are not limited to the followingcompounds:

III. Synthesis of Compounds

The compounds disclosed herein can be synthesized generally as disclosedherein, and using methods available in the art. For example, thefollowing general scheme represents the synthesis of an adenosinecompound:

General Scheme for Synthesis of an Adenosine N6-Modified Compound

General Scheme for Synthesis Guanosine O6-Modified Compounds

General Scheme for Synthesis Guanosine 8-Oxo-Modified Compounds

General Scheme for Synthesis Uridine 5-HOMe-Modified Compounds

General Scheme for Synthesis Cytidine 5-HOMe-Modified Compounds

General Scheme for Synthesis Cytidine N4-Modified Compounds

Additional details are provided in the Examples section.

IV. Methods of Use of Compounds According to the Invention

The nucleoside compounds disclosed herein can be used in for a varietyof purposes in DNA sequencing technology. Polymerases used inconjunction with the compounds according to the invention may be nativepolymerases or modified polymerases. Polymerases include DNA and non-DNApolymerases. For example, polymerases for use with the compoundsaccording to the invention include without limitation reversetranscriptase, terminal transferase, and DNA polymerase. Among DNApolymerases, preferred embodiments include Taq DNA polymerase,Klenow(-exo-) DNA polymerase, Bst DNA polymerase, Vent(-exo-) DNApolymerase, Pfu(-exo-) DNA polymerase, and DeepVent(-exo-) DNApolymerase. Modified polymerases include without limitation TaqFS DNApolymerase, ThermoSequenase DNA polymerase, ThermoSequenase II DNApolymerase, Therminator DNA polymerase, Therminator II DNA polymerase,and Vent(-exo-) A488L DNA polymerase. Preferably, compounds according tothe invention are incorporated at levels equal to or near theincorporation levels of naturally-occurring nucleotides, thus resultingin no bias against the compounds according to the invention. Even morepreferably, compounds according to the invention are compatible withcommercially-available polymerases.

In a preferred embodiment, methods according to the invention include amethod of determining the sequence of a target nucleic acid comprising(i) adding a target nucleic acid to a Sanger or Sanger-type sequencingapparatus, (ii) adding one or more compounds according to the inventionto the sequencing apparatus, with the proviso that where more than onetype of base is present, each base is attached to a differentfluorophore; (iii) adding a complementary primer and a polymeraseenzyme, (iv) performing a polymerase reaction to incorporate at leastone of the compounds of step (ii) into a growing nucleic acid strand,and (v) analyzing the result of the Sanger sequencing reaction withfluorescence sequencing instrumentation or by pulsed multilineexcitation fluorescence, wherein steps (i)-(iii) can be performed in anyorder.

In a preferred embodiment, incorporation of at least one compoundaccording to step (iv) is followed by termination of strand growth at anefficiency of from about 90% to about 100%. Alternatively, theincorporation of at least one compound according to step (iv) occurs atabout 70% to about 100% of the efficiency of incorporation of a nativesubstrate with the same base in the polymerase reaction, or morepreferably at about 85% to about 100%.

Methods according to the invention also include a method ofincorporating a non-naturally occurring component into a nucleic acidcomprising: (i) adding a target nucleic acid to a sequencing apparatus;(ii) adding one or more compounds according to the invention to thesequencing apparatus, with the proviso that where more than one type ofbase is present, each base is attached to a different fluorophore; (iii)adding a polymerase enzyme; and (iv) performing a polymerase reaction toincorporate at least one of the compounds of step (ii) into a growingnucleic acid strand, wherein steps (i)-(iii) can be performed in anyorder. The method can further comprise (v) analyzing the result of thepolymerase chain reaction for incorporation of at least one compoundfrom step (ii).

An alternative embodiment of the invention is a method of terminatingnucleic acid synthesis comprising the step of placing a 3′-OHunprotected nucleotide or nucleoside according to the invention in theenvironment of a polymerase and allowing incorporation of the 3′-OHunprotected nucleotide or nucleoside into a nucleic acid. Preferredembodiments of the method have an efficiency of termination uponincorporation of the 3′-OH unprotected nucleotide or nucleoside rangingfrom about 90% to about 100%; with the efficiency of incorporation ofthe 3′-OH unprotected nucleotide or nucleoside ranging from about 70% toabout 100% compared to the efficiency of incorporation of anaturally-occurring nucleotide or nucleoside with the same base.

Methods of performing Sanger or Sanger-type sequencing comprisingaddition of a compound according to the invention to a Sanger orSanger-type sequencing method are also encompassed. A method ofperforming mini-sequencing or minisequencing-type sequencing comprisingaddition of a compound according to the invention to a mini-sequencingor minisequencing-type sequencing method is within the scope of theinvention.

PME Detector

In one embodiment, a pulsed-multiline excitation (“PME”) for color-blindfluorescence detection can be used as described in US 2003/0058440published Mar. 27, 2003, or PCT WO 031 021212. published Mar. 13, 2003.This technology provides fluorescence detection with application forhigh-throughput identification of informative SNPs, for more accuratediagnosis of inherited disease, better prognosis of risksusceptibilities, or identification of sporadic mutations. The PMEtechnology has two main advantages that significantly increasefluorescence sensitivity: (1) optimal excitation of all fluorophores inthe genomic assay and (2) “color-blind” detection, which collectsconsiderably more light than standard wavelength resolved detection.This technology differs significantly from DNA sequencinginstrumentation which features single source excitation and colordispersion for DNA sequence identification. The technology can be usedin clinical diagnostics, forensics, and general sequencing methodologiesand will have the capability, flexibility, and portability of targetedsequence variation assays for a large majority of the population.

In one embodiment, an apparatus and method for use in high-throughputDNA sequence identification is used. A pulse-multiline excitationapparatus for analyzing a sample containing one or more fluorescentspecies is used, comprising: one or more lasers configured to emit twoor more excitation lines, each excitation line having a differentwavelength; a timing circuit coupled to the one or more lasers andconfigured to generate the two or more excitation lines sequentiallyaccording to a timing program to produce time-correlated fluorescenceemission signals from the sample; a non-dispersive detector positionedto collect the time-correlated fluorescence emission signals emanatingfrom the sample; and an analyzer coupled to the detector and configuredto associate the time-correlated fluorescence emission signals with thetiming program to identify constituents of the sample.

The detector and the analyzer may be integral. In one embodiment, thetwo or more excitation lines intersect at the sample, or the two or moreexcitation lines may be configured so that they do not intersect in thesample. The two or more excitation lines may be coaxial.

In one embodiment, the apparatus may further comprise an assembly of oneor more prisms in operative relation with the one or more lasers andconfigured to render radiation of the two or more excitation linessubstantially colinear and/or coaxial.

The apparatus may have a plurality of excitation lines, for example 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more excitationlines having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 ormore excitation wavelengths, respectively. The sample may be comprised aplurality of vessels such as capillaries, for example in 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, up to 20, up to 24, up to 28, upto 36, up to 48, up to 64, up to 96, up to 384 or more capillaries. Asheath flow cuvette may be used.

The timing program may comprise a delay between the firing of each laserof between about 10 fs and about 5 s, between about 1 ms and about 100ms, or between about 50 ps and about 500 ps. One or more of theexcitation lines is pulsed. The pulsed excitation line may be controlledby TTL logic or by mechanical or electronic means. In one embodiment,the apparatus may generate a sequence of discrete excitation lines thatare time-correlated with the fluorescence emission signals from thesample.

The lasers may independently comprise a diode laser, a semiconductorlaser, a gas laser, such as an argon ion, krypton, or helium-neon laser,a diode laser, a solid-state laser such as a Neodymium laser which willinclude an ion-gain medium, such as YAG and yttrium vanadate (YVO₄), ora diode pumped solid state laser. Other devices, which produce light atone or more discrete excitation wavelengths, may also be used in placeof the laser. The laser may further comprise a Raman shifter in operablerelation with at least one laser beam. In one embodiment of theinvention, the excitation wavelength provided by each laser is opticallymatched to the absorption wavelength of each fluorophore.

The detector may comprise a charged couple device, a photomultipliertube, a silicon avalanche photodiode or a silicon PIN detector. Thefootprint of the device is preferably small, such as less than 4 ft×4ft×2 ft, less than 1 ft×1 ft×2 ft, and could be made as small as 1 in×3in×6 in.

Another aspect comprises a method of identifying sample componentscomprising: (a) preparing a sample comprising sample components, a firstdye and a second dye; (b) placing the sample in the beam path of a firstexcitation line and a second excitation line; (c) sequentially firingthe first excitation line and the second excitation line; (d) collectingfluorescence signals from the samples as a function of time; and (e)sorting the fluorescence by each excitation line's on-time window,wherein the sample components are identified. It is an aspect of theinvention that the fluorescence signals are collected from discrete timeperiods in which no excitation line is incident on the sample, the timeperiods occurring between the firing of the two excitation lines. Thistechnique is known as “looking in the dark.” Yet another aspect is thatthe absorption maximum of the first dye substantially corresponds to theexcitation wavelength of the first excitation line. The absorptionmaximum of the second dye may also substantially corresponds to theexcitation wavelength of the second excitation line. In yet anotheraspect there is a third and fourth dye and a third and fourth excitationline, wherein the absorption maxima of the third and fourth dyessubstantially correspond to the excitation wavelengths of the third andfour excitation lines, respectively. Similarly, there may be 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16 or more dyes wherein the absorption maximaof the dyes substantially corresponds to excitation wavelengths of a5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, or moreexcitation lines, respectively. The dyes may be a xanthene, fluorescein,rhodamine, BODIPY, cyanine, coumarin, pyrene, phthalocyanine,phycobiliprotein, Alexa, squariane dyes, or some other suitable dye.

In one embodiment, sample components enable the determination of SNPs.The method may be for the high-throughput identification of informativeSNPs. The SNPs may be obtained directly from genomic DNA material, fromPCR amplified material, or from cloned DNA material and may be assayedusing a single nucleotide primer extension method. The single nucleotideprimer extension method may comprise using single unlabeled dNTPs,single labeled dNTPs, single 3′-modified dNTPs, single base-modified3′-dNTPs, single alpha-thio-dNTPs or single labeled2′,3′-dideoxynucleotides. The mini-sequencing method may comprise usingsingle unlabeled dNTPs, single labeled dNTPs, single 3′-modified dNTPs,single base-modified 3′-dNTPs, single alpha-thio-dNTPs or single labeled2′,3′-dideoxynucleotides. The SNPs may be obtained directly from genomicDNA material, from PCR amplified material, or from cloned DNA materials.

Also envisioned are methods for detecting nucleic acids. Nucleic acidsmay be detected in situ or in various gels, blots, and similar methodsfor detecting nucleic acids, such as disclosed in U.S. Pat. No.7,125,660, which is incorporated herein by reference.

EXAMPLES Example 1 dA Compounds Synthesis of N⁶-benzyl-2′-deoxyadenosinetriphosphate (WW2p062)

N⁶-tert-Butyloxycarbonyl-N⁶-benzyl-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyadenosine(dA.n1)

NaH (18 mg, 0.75 mmol, dry) was added to a solution of compound dA.07(400 mg, 0.58 mmol) in anhydrous DMF (5 mL) at 0° C. and stirred for 30minutes. A solution of benzyl bromide (149 mg, 0.87 mmol) in anhydrousDMF (2.5 mL) was added dropwise.

The mixture was gradually warmed to room temperature and stirred for twohours. DMF was removed in vacuo, and the residue was dissolved in ethylacetate (60 mL), washed twice with saturated NH₄Cl solution (40 mL each)and once with water (40 mL). The combined aqueous layer was extractedwith ethyl acetate (10 mL), and the combined organic layer was driedover Na₂SO₄, concentrated in vacuo, and purified by silica gel columnchromatography to yieldN⁶-tert-butyloxycarbonyl-N⁶-benzyl-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyadenosinedA.n1 (398 mg, 86%) as a viscous oil.

¹H NMR (400 MHz, CDCl₃): δ 8.72 (s, 1H, H-8), 8.32 (s, 1H, H-2), 7.39(m, 2H, Ph-H), 7.25 (m, 2H, Ph-H), 7.18 (m, 1H, Ph-H), 6.49 (t, 1H,J=6.4 Hz, H-1′), 5.28 (s, 2H, Ph-CH₂), 4.62 (m, 1H, H-3′), 4.01 (m, 1H,H-4′), 3.85 (dd, 1H, J=4.4 and 11.2 Hz, H-5′a), 3.77 (dd, 1H, J=3.4 and11.2 Hz, H-5′b), 2.61 (m, 1H, H-2′a), 2.43 (m, 1H, H-2′b), 1.65 (s, 9H,(CH₃)₃CO), 0.96 (s, 18H, (CH₃)₃CSi), 0.08 (2 s, 12H, (CH₃)₂Si).

N⁶-Benzyl-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyadenosine (dA.n2)

Silica gel 60 (3.76 g, 100-200 mesh, activated by heating to 70-80° C.under reduced pressure for hours) was added to a solution of compounddA.n1 (376 mg, 0.56 mmol) in CH₂Cl₂ (20 mL), and the mixture wasevaporated in vacuo to dryness. The residue was heated to 70-80° C.under reduced pressure for two days, washed three times with methanol(30 mL each), and filtered using a buchi funnel. The combined filtratewas concentrated in vacuo and purified by silica gel columnchromatography to yieldN⁶-benzyl-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyadenosine dA.n2(305 mg, 95%) as a yellow foam.

¹H NMR (400 MHz, CDCl₃): δ 8.41 (s, 1H, H-8), 8.07 (s, 1H, H-2), 7.38(m, 2H, Ph-H), 7.33 (m, 2H, Ph-H), 7.28 (m, 1H, Ph-H), 6.45 (t, 1H,J=6.4 Hz, H-1′), 6.12 (br s, 1H, 6-NH), 4.87 (br s, 2H, Ph-CH₂), 4.62(m, 1H, H-3′), 4.01 (m, 1H, H-4′), 3.87 (dd, 1H, J=4.2 and 11.2 Hz,H-5′a), 3.77 (dd, 1H, J=3.2 and 11.2 Hz, H-5′b), 2.64 (m, 1H, H-2′a),2.44 (m, 1H, H-2′b), 0.91 (s, 18H, (CH₃)₃CSi), 0.09 (2 s, 12H,(CH₃)₂Si-).

N⁶-Benzyl-2′-deoxyadenosine (dA.n3)

A solution of n-Bu₄NF (335 mg, 1.28 mmol) in THF (2.5 mL) was added to asolution of compound dA.n2 (292 mg, 0.51 mmol) in THF (6 mL) at 0° C.The reaction mixture was gradually warmed to room temperature andstirred for two hours. Silica gel 60 (1 g) was added, and the mixturewas evaporated in vacuo to dryness. The residue was purified by silicagel column chromatography to yield N⁶-benzyl-2′-deoxyadenosine dA.n3(173 mg, 99%) as a white foam.

¹H NMR (400 MHz, CD₃OD): δ 8.30 (s, 1H, H-8), 8.25 (s, 1H, H-2), 7.36(m, 2H, Ph-H), 7.31 (m, 2H, Ph-H), 7.24 (m, 1H, Ph-H), 6.42 (dd, 1H,J=6.0 and 7.9 Hz, H-1′), 4.81 (br s, 2H, Ph-CH₂), 4.57 (m, 1H, H-3′),4.06 (m, 1H, H-4′), 3.83 (m, 1H, J=2.9 and 12.3 Hz, H-5′a), 3.73 (dd,1H, J=3.3 and 12.3 Hz, H-5′b), 2.79 (m, 1H, H-2′a), 2.40 (m, 1H, H-2′b).

N⁶-Benzyl-2′-deoxyadenosine-5′-triphosphate (WW2p062)

POCl₃ (22 μL, 0.24 mmol) was added to a solution of compound dA.10a (42mg, 0.12 mmol) in trimethylphosphate (0.5 mL) and maintained at minus20-30° C. for two hours. A solution of bis-tri-n-butylammoniumpyrophosphate (285 mg, 0.6 mmol) and tri-n-butylamine (120 μl) inanhydrous DMF (1.2 mL) was added. After five minutes of stirring,triethylammonium bicarbonate buffer (1 M, pH 7.5; 10 mL) was added. Thereaction was stirred at room temperature for one hour and thenlyophilized to dryness. The residue was dissolved in water (10 mL),filtered, and purified by anion exchange chromatography using a QSepharose FF column (2.5×20 cm) with a linear gradient of NH₄HCO₃ (50 mMto 500 mM in 300 minutes) at a flow rate of 4.5 mL/min. The fractionscontaining triphosphate were combined and lyophilized to giveN⁶-benzyl-2′-deoxyadenosine-5′-triphosphate WW2p062 (24 mg, 32%) as awhite fluffy solid.

¹H NMR (400 MHz, D₂O): δ 8.43 (s, 1H, H-8), 8.20 (s, 1H, H-2), 7.39-7.30(m, 5H, Ph-H), 6.50 (t, 1H, J=6.4 Hz, H-1′), 4.85 (s, 2H, Ph-CH₂), 4.31(s, 1H, H-4′), 4.22 (m, 2H, H-5′a and H-5′b), 2.82 (m, 1H, H-2′a), 2.62(m, 1H, H-2′b);

³¹P NMR (162 MHz, D₂O): δ −5.72 (d, J=15.9 Hz), −10.78 (d, J=15.4 Hz),−19.16 (t, J=14.9 Hz);

ToF-MS (ESI): For the molecular ion C₁₇H₂₀N₅O₁₂P₃Na [M−2H+Na]⁻, thecalculated mass was 602.0219, and the observed mass was 602.0363.

Synthesis of 6-FAM labeledN⁶-[4-(3-amino-1-propyl)benzyl]-2′-deoxyadenosine triphosphate (WW2p085)

N⁶-tert-Butyloxycarbonyl-N⁶-(4-iodobenzyl)-3′,5 2′-deoxyadenosine(dA.n4)

NaH (23 mg, 0.94 mmol, dry) was added to a solution of compound dA.07(500 mg, 0.72 mmol) in anhydrous DMF (6.5 mL) at 0° C. and stirred for30 minutes. A solution of 4-Iodobenzyl bromide (322 mg, 1.08 mmol) inanhydrous DMF (2.5 mL) was added dropwise. The mixture was graduallywarmed to room temperature and stirred for two hours. DMF was removed invacuo, and the residue was dissolved in ethyl acetate (60 mL), washedtwice with saturated NH₄Cl solution (40 mL each) and once with water (40mL). The combined aqueous layer was extracted with ethyl acetate (10mL), and the combined organic layer was dried over Na₂SO₄, concentratedin vacuo, and purified by silica gel column chromatography to yieldN⁶-tert-butyloxycarbonyl-N⁶-(4-iodobenzyl)-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyadenosinedA.n4 (565 mg, 99%) as a viscous oil.

¹H NMR (400 MHz, CDCl₃): δ 8.71 (s, 1H, H-8), 8.33 (s, 1H, H-2), 7.58(d, 2H, J=8.2 Hz, Ph-H), 7.17 (d, 2H, J=8.2 Hz, Ph-H), 6.49 (t, 1H,J=6.4 Hz, H-1′), 5.20 (s, 2H, Ph-CH₂), 4.62 (m, 1H, H-3′), 4.02 (m, 1H,H-3′), 3.86 (dd, 1H, J=4.2 and 11.2 Hz, H-5′a), 3.78 (dd, 1H, J=3.2 and11.2 Hz, H-5′b), 2.63 (m, 1H, H-2′a), 2.45 (m, 1H, H-2′b), 1.42 (s, 9H,(CH₃)₃CO), 0.92 (s, 18H, (CH₃)₃CSi), 0.08 (2 s, 12H, (CH₃)₂Si-).

N⁵-(4-Iodobenzyl)-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyadenosine(dA.n5)

Silica gel 60 (6.00 g, 100-200 mesh, activated by heating to 70-80° C.under reduced pressure for hours) was added to a solution of compounddA.n4 (565 mg, 0.71 mmol) in CH₂Cl₂ (20 mL), and the mixture wasevaporated in vacuo to dryness. The residue was heated to 70-80° C.under reduced pressure for two days, washed three times with methanol(30 mL each), and filtered using a buchi funnel. The combined filtratewas concentrated in vacuo and purified by silica gel columnchromatography to yieldN⁶-(4-iodobenzyl)-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyadenosinedA.n5 (489 mg, 99%) as a yellow foam.

¹H NMR (400 MHz, CDCl₃): δ 8.38 (s, 1H, H-8), 8.06 (s, 1H, H-2), 7.63(d, 2H, J=8.2 Hz, Ph-H), 7.11 (d, 2H, J=8.2 Hz, Ph-H), 6.45 (t, 1H,J=6.4 Hz, H-1′), 6.34 (t, 1H,

6-NH), 4.81 (br s, 2H, Ph-CH₂), 4.61 (m, 1H, H-3′), 4.00 (m, 1H, H-4′),3.85 (dd, 1H, J=4.2 and 11.2 Hz, H-5′a), 3.76 (dd, 1H, J=3.2 and 11.2Hz, H-5′b), 2.64 (m, 1H, H-2′a), 2.44 (m, 1H, H-2′b), 0.91 (s, 18H,(CH₃)₃CSi), 0.09 (2 s, 12H, (CH₃)₂Si-).

N⁶-(4-Iodobenzyl)-2′-deoxyadenosine (dA.n6)

A solution of n-Bu₄NF (282 mg, 1.08 mmol) in THF (1.0 mL) was added to asolution of compound dA.n5 (300 mg, 0.43 mmol) in THF (1.2 mL) at 0° C.The reaction mixture was gradually warmed to room temperature andstirred for two hours. Silica gel 60 (1 g) was added, and the mixturewas evaporated in vacuo to dryness. The residue was purified by silicagel column chromatography to yield N⁶-(4-iodobenzyl)-2′-deoxyadenosinedA.n6 (266 mg, 98%) as a white foam.

¹H NMR (400 MHz, DMSO-d₆): δ 8.48 (br s, 1H, D₂O exchangeable, 6-NH),8.40 (s, 1H, H-8), 8.27 (s, 1H, H-2), 7.68 (d, 2H, J=8.0 Hz, Ph-H), 7.17(d, 2H, J=8.0 Hz, Ph-H), 6.39 (t, 1H, J=6.4 Hz, H-1′), 5.34 (d, 1H, D₂Oexchangeable, 3′-OH), 5.22 (t, 1H, D₂O exchangeable, 5′-OH), 4.68 (br s,2H, Ph-CH₂), 4.44 (m, 1H, H-4′), 3.91 (m, 1H, H-3′), 3.64 (m, 1H,H-5′a), 3.55 (m, 1H, H-5′b), 2.76 (m, 1H, H-2′a), 2.31 (m, 1H, H-2′b).

N⁶-[4-(3-trifluoroacetamido-1-propynyl)benzyl]-2′-deoxyadenosine (dA.n7)

A solution of compound dA.n6 (266 mg, 0.57 mmol),N-propargyltrifluoroacetamide (260 mg, 1.72 mmol), CuI (22 mg, 0.11mmol), tetrakis(triphenylphosphine)-palladium(0) (65 mg, 0.06 mmol), andEt₃N (160 μL, 1.14 mmol) in anhydrous DMF (2.1 mL) was stirred at roomtemperature for 4.5 hours. The mixture was concentrated in vacuo andpurified by silica gel column chromatography to yieldN⁶-[4-(3-trifluoroacetamido-1-propynyl)-benzyl]-2′-deoxyadenosine dA.n7(268 mg, 94%) as a waxy solid.

¹H NMR (400 MHz, DMSO-d₆): δ 10.05 (br m, 1H, D₂O exchangeable, NH),8.46 (br m, 1H, D₂O exchangeable, NH), 8.37 (s, 1H, H-8), 8.19 (s, 1H,H-2), 7.37 (d, 2H, J=8.2 Hz, Ph-H), 7.32 (d, 2H, J=8.2 Hz, Ph-H), 6.35(dd, 1H, J=6.4 and 7.5 Hz, H-1′), 5.31 (d, 1H, D₂O exchangeable, 3′-OH),5.19 (t, 1H, D₂O exchangeable, 5′-OH), 4.70 (br s, 2H, Ph-CH₂), 4.41 (m,1H, H-3′), 4.26 (d, 2H, J=4.3 Hz, CH₂) 3.88 (m, 1H, H-4′), 3.61 (m, 1H,H-5′a), 3.53 (m, 1H, H-5′b), 2.73 (m, 1H, H-2′a), 2.25 (m, 1H, H-2′b).

N⁶-[4-(3-Amino-1-propyl)benzyl]-2′-deoxyadenosine-5′-triphosphate(dA.n8)

POCl₃ (16 μL, 0.17 mmol) was added to a solution of compound dA.n7 (56mg, 0.11 mmol) and proton sponge (37 mg, 0.17 mmol) intrimethylphosphate (0.5 mL) and maintained at minus 20-30° C. for twohours. A solution of bis-tri-n-butylammonium pyrophosphate (261 mg, 0.55mmol) and tri-n-butylamine (110 μL) in anhydrous DMF (1.1 mL) was added.After five minutes of stirring, triethylammonium bicarbonate buffer (1M, pH 7.5; 10 mL) was added. The reaction was stirred for one hour atroom temperature, followed by the dropwise addition of concentratedammonium hydroxide (10 mL, 27%) at 0° C. The mixture was stirred for anadditional hour at room temperature and then lyophilized to dryness. Theresidue obtained was dissolved in water (10 mL), filtered, and purifiedby anion exchange chromatography using a Q Sepharose FF Column (2.5×20cm) with a linear gradient of NH₄HCO₃ (50 mM to 500 mM in 300 minutes)at a flow rate of 4.5 mL/min. The fractions containing triphosphate werecombined and lyophilized to give triphosphate dA.n8 (63 mg, 84%) as awhite fluffy solid.

¹H NMR (400 MHz, D₂O): δ 8.41 (s, 1H, H-8), 8.19 (s, 1H, H-2), 7.38-7.26(m, 4H, Ph-H), 6.47 (dd, 1H, J=5.5 and 6.6 Hz, H-1′), 4.30 (s, 1H,H-4′), 4.21 (m, 2H, H-5′a and H-5′b), 3.63 (s, 2H, CH₂), 2.79 (m, 1H,H-2′a), 2.60 (m, 1H, H-2′b).

³¹P NMR (162 MHz, D₂O): δ −5.80 (d, J=20.1 Hz), −10.94 (d, J=19.3 Hz),−21.59 (t, J=19.3 Hz);

ToF-MS (ESI): For the molecular ion C₂₀H₂₃N₆O₁₂P₃Na [M−2H+Na]⁻, thecalculated mass was 655.0485, and the observed mass was 655.0758.

6-FAM labeledN⁶-[4-(3-Amino-1-propyl)benzyl]-2′-deoxyadenosine-5′-triphosphate(WW2p085)

A solution of 6-FAM-SE (3.5 mg, 7.35 μmol) in anhydrous DMSO (70 μL) wasadded to a solution of triphosphate dA.18a (3.5 μmol) in Na₂CO₃/NaHCO₃buffer (0.1 M, pH 9.2; 600 μL) and incubated at room temperature for onehour. The reaction was purified by reverse-phase HPLC using a PerkinElmer OD-300 C₁₈ column (4.6×250 mm) to yield the 6-FAM labeledtriphosphate WW2p085. Mobile phase: A, 100 mM triethylammonium acetate(TEAA) in water (pH 7.0); B, 100 mM TEAA in water/CH₃CN (30:70). Elutionwas performed with a linear gradient of 5-20% B for 20 minutes and then20-90% B for 20 minutes. The concentration of WW2p085 was estimated byadsorption spectroscopy using the extinction coefficient of the 6-FAMdye (i.e., 68,000 at 494 nm).

ToF-MS (ESI): For the molecular ion C₄₁H₃₆N₆O₁₈P₃ [M+H]⁺, the calculatedmass was 993.1299, and the observed mass was 993.1520.

Synthesis of 6-FAM labeledN⁶-{1-[4-(3-amino-1-propynyl)phenyl]ethyl}-2′-deoxyadenosinetriphosphate (WW2p093)

3′,5′-O-Bis-tert-butyldimethylsilyl-2′-deoxyinosine (dA.n9)¹

¹The exact procedure can be found in: Kiselyov, A. S.; Steinbrecher, T.;Harvey, R. G. (1995) “Synthesis of the Fjord-region cis- and trans-AminoTriol Derivatives of the carcinogenic Hydrocarbon Benzo[g]chrysene andUtilization for the Synthesis of a Deoxyadenosine Adduct Linked to theN6-Amino Group” J. Org. Chem., 60: 6129-6134.

A solution of TBSCl (1.91 g, 12.67 mmol) was added to a solution of2′-deoxyinosine (1.00 g, 3.96 mmol) and imidiazole (1.73 g, 25.34 mmol)in anhydrous DMF (3 mL) at 0° C. under nitrogen atmosphere. The reactionmixture was gradually warmed to room temperature and stirred for 12hours. The mixture was then concentrated in vacuo, dissolved in CH₂Cl₂(100 mL), washed twice with water (50 mL), dried over anhydrous Na₂SO₄,concentrated in vacuo, and purified by silica gel chromatography toyield 3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyinosine dA.n9 (1.58 g,83%) as a white powder.

¹H NMR (400 MHz, DMSO-d₆): δ 12.37 (br s, 1H, D₂O exchangeable, NH),8.25 (s, 1H, H-8), 8.04 (d, 1H, J=3.6 Hz, H-2), 6.29 (t, 1H, J=6.6 Hz,H-1′), 4.59 (m, 1H, H-3′), 3.84 (m, 1H, H-4′), 3.74 (m, 1H, H-5′a), 3.66(m, 1H, H-5′b), 2.76 (m, 1H, H-2′a), 2.30 (m, 1H, H-2′b), 0.89 (s, 9H,(CH₃)₃CSi), 0.85 (s, 9H, (CH₃)₃CSi), 0.11 (s, 6H, (CH₃)₂Si), 0.02 (2 s,6H, (CH₃)₂Si).

O⁶-(2-Mesitylenesulfonyl)-3′,5′-bis-O-tert-butyldimethylsilyl-2′-deoxyinosine(dA.n10)¹

2-Mesitylenesulfonyl chloride (0.70 g, 2.12 mmol), Et₃N (0.42 mL, 3.07mmol), and DMAP (16 mg, 0.13 mmol) were added to a solution of dA.n9(1.02 g, 2.12 mmol) in anhydrous CH₂Cl₂ (15 mL). The reaction mixturewas stirred at room temperature for 1.5 hours, then diluted with ethylether (50 mL). The ether solution was washed twice with a saturatedsolution of NaHCO₃ (25 mL each) and then with brine (25 mL). The organiclayer was dried over Na₂SO₄, concentrated in vacuo, and purified bysilica gel chromatography to yieldO⁶-(2-mesitylenesulfonyl)-3′,5′-bis-O-tert-butyldimethylsilyl-2′-deoxyinosinedA.n10 (279 mg, 20%).

¹H NMR (400 MHz, CDCl₃): δ 8.55 (s, 1H, H-8), 8.38 (s, 1H, H-2), 6.99(s, 2H, Ph-H), 6.48 (t, 1H, J=6.4 Hz, H-1′), 4.61 (m, 1H, H-3′), 4.03(m, 1H, H-4′), 3.85 (m, 1H, H-5′a), 3.76 (m, 1H, H-5′b), 2.77 (s, 6H,CH₃), 2.61 (m, 1H, H-2′a), 2.43 (m, 1H, H-2′b), 2.32 (s, 3H, CH₃), 0.91(s, 9H, (CH₃)₃CSi), 0.89 (s, 9H, (CH₃)₃CSi), 0.09 (s, 6H, (CH₃)₂Si),0.08 (2 s, 6H, (CH₃)₂Si).

N⁶-[1-(4-Iodophenyl)ethyl]-3′,5′-bis-O-tert-butyldimethylsilyl-2′-deoxyadenosine(dA.n11)

A solution of 1-(4-iodophenyl)ethylamine (312 mg, 1.26 mmol) inanhydrous 1,4-dioxane (1 mL) was added to a solution of dA.n10 (279 mg,0.42 mmol) in anhydrous 1,4-dioxane (2 mL) containing molecular sieves(4 Å, 8-12 Mesh, 0.75 g) at room temperature under nitrogen atmosphere.The mixture was then stirred at 50° C. for 18 hours. The solvent wasremoved in vacuo, and the crude product was purified by silica gelcolumn chromatography to yieldN⁶-[1-(4-iodophenyl)ethyl]-3′,5′-bis-O-tert-butyldimethylsilyl-2′-deoxyadenosinedA.n11 (263 mg, 88%, 1:1 mixture of diastereoisomers) as a white foam.

¹H NMR (400 MHz, CDCl₃) for diastereoisomers: δ 8.32 (s, 1H, H-8), 8.08(s, 1H, H-2), 7.61 (m, 2H, Ph-H), 7.15 (m, 2H, Ph-H), 6.42 (t, 1H, J=6.4Hz, H-1′), 6.20 (br m, 1H, NH), 5.50 (br s, 1H, Ph-CH), 4.59 (m, 1H,H-3′), 3.99 (m, 1H, H-4′), 3.85 (m, 1H, H-5′a), 3.77 (m, 1H, H-5′b),2.60 (m, 1H, H-2′a), 2.42 (m, 1H, H-2′b), 1.59 (d, 3H, J=7.0 Hz, CH₃),0.90 (s, 18H, (CH₃)₃CSi), 0.08 (s, 12H, (CH₃)₂Si);

¹³C NMR (100 MHz, MeOH-d₄) for diastereoisomers: δ 153.78 (C), 151.94(CH), 143.78/143.71 (C), 138.36 (CH), 137.57 (CH), 128.17/128.16 (CH),119.99 (C), 92.41 (C), 87.81/87.79 (CH), 84.28 (CH), 71.78/71.74 (CH),62.72/62.68 (CH₂), 49.40 (br, CH), 41.31 (CH₂), 25.96 (CH₃), 25.75(CH₃), 22.64 (CH₃), 18.41 (C), 17.99 (C), −4.66 (CH₃), −4.82 (CH₃),−5.39 (CH₃), −5.48 (CH₃).

N⁶-[1-(4-Iodophenyl)ethyl]-2′-deoxyadenosine (dA.n12)

A solution of n-Bu₄NF (409 mg, 1.30 mmol) in THF (3 mL) was added to asolution of compound dA.n11 (263 mg, 0.37 mmol) in THF (5 mL) at 0° C.The reaction mixture was gradually warmed to room temperature andstirred for 30 minutes, then concentrated in vacuo to dryness. Theresidue was purified by silica gel column chromatography to yieldN⁶-[1-(4-iodophenyl)ethyl]-2′-deoxyadenosine dA.n12 (164 mg, 93% 1:1mixture of diastereoisomers) as a waxy solid.

¹H NMR (400 MHz, DMSO-d_(s)) for diastereomers: δ 8.35 (s, 1H, H-8),8.32 (br s, 1H, D₂O exchangeable, NH), 8.13 (s, 1H, H-2), 7.62 (d, 2H,J=8.2 Hz, Ph-H), 7.22 (d, 2H, 2H, J=8.2 Hz, Ph-H), 6.32 (m, 1H, H-1′),5.41 (br, 1H, Ph-CH), 5.31 (d, 1H, D₂O exchangeable, 3′-OH), 5.19 (m,1H, D₂O exchangeable, 5′-OH), 4.35 (m, 1H, H-4′), 3.85 (m, 1H, H-4′),3.58 (m, 1H, H-5′a), 3.48 (m, 1H, H-5′b), 2.68 (m, 1H, H-2′a), 2.22 (m,1H, H-2′b), 1.49 (d, 3H, J=7.0 Hz, CH₃).

N⁶-{1-[4-(3-Trifluoroacetamido-1-propynyl)phenyl]ethyl}-2′-deoxyadenosine(dA.n13)

A solution of compound dA.n12 (70 mg, 0.145 mmol),N-propargyltrifluoroacetamide (66 mg, 0.44 mmol), CuI (5.5 mg, 0.03mmol), tetrakis(triphenylphosphine)-palladium(0) (17 mg, 0.015 mmol),and Et₃N (41 μL, 0.29 mmol) in anhydrous DMF (2.2 mL) was stirred atroom temperature for 5.5 hours. The mixture was concentrated in vacuoand purified by silica gel column chromatography to yieldN⁶-{1-[4-(3-trifluoroacetamido-1-propynyl)phenyl]ethyl}-2′-deoxyadenosinedA.n13 (63 mg, 86%, 1:1 mixture of diastereoisomers) as a waxy solid.

¹H NMR (400 MHz, DMSO-d₆) for diastereomers: δ 10.05 (t, 1H, J=5.4 Hz,D₂O exchangeable, NH), 8.36 (s, 1H, H-8), 8.34 (br s, 1H, D₂Oexchangeable, NH), 8.15 (s, 1H, H-2), 7.43 (d, 2H, J=8.2 Hz, Ph-H), 7.36(d, 2H, 2H, J=8.2 Hz, Ph-H), 6.33 (dd, 1H, J=6.4 and 7.5, Hz, H-1′),5.49 (br, 1H, Ph-CH), 5.30 (d, 1H, D₂O exchangeable, 3′-OH), 5.10 (m,1H, D₂O exchangeable, 5′-OH), 4.39 (m, 1H, H-3′), 4.25 (d, 2H, J=5.4 Hz,CH₂), 3.87 (m, 1H, H-3′), 3.59 (m, 1H, H-5′a), 3.51 (m, 1H, H-5′b), 2.72(m, 1H, H-2′a), 2.24 (m, 1H, H-2′b), 1.52 (d, 3H, J=7.0 Hz, CH₃);

N⁶-{1-[4-(3-Amino-1-propynyl)phenyl]ethyl}-2′-deoxyadenosine-5′-triphosphate(dA.n14)

POCl₃ (14 μL, 0.15 mmol) was added to a solution of compound dA.n14 (51mg, 0.1 mmol) and proton sponge (32 mg, 0.15 mmol) in trimethylphosphate(0.5 mL) and maintained at minus 20-30° C. for two hours. A solution ofbis-tri-n-butylammonium pyrophosphate (237 mg, 0.5 mmol) andtri-n-butylamine (100 μL) in anhydrous DMF (1.0 mL) was added. Afterfive minutes of stirring, triethylammonium bicarbonate buffer (1 M, pH7.5; 10 mL) was added. The reaction was stirred for one hour at roomtemperature, followed by the dropwise addition of concentrated ammoniumhydroxide (10 mL, 27%) at 0° C. The mixture was stirred for anadditional hour at room temperature and then lyophilized to dryness. Theresidue obtained was dissolved in water (10 mL), filtered, and purifiedby anion exchange chromatography using a Q Sepharose FF column (2.5×20cm) with a linear gradient of NH₄HCO₃ (50 mM to 500 mM in 300 minutes)at a flow rate of 4.5 mL/min. The fractions containing triphosphate werecombined and lyophilized to give triphosphate dA.n14 (60 mg, 86%, 1:1mixture of diastereoisomers) as a white fluffy solid.

¹H NMR (400 MHz, D₂O) for diastereoisomers: δ 8.41 (s, 1H, H-8), 8.14 (2s, 1H, H-2), 7.38 (m, 4H, Ph-H), 6.46 (m, 1H, H-1), 5.32 (br, 1H,Ph-CH), 4.30 (s, 1H, H-3′), 4.20 (m, 2H, H-5′a and H-5′b), 3.61 (s, 2H,CH₂), 2.78 (m, 1H, H-2′a), 2.59 (m, 1H, H-2′b), 1.60 (d, 3H, J=6.9 Hz,CH₃);

³¹P NMR (162 MHz, D₂O): δ −6.02 (d, J=19.4 Hz), −11.19 (d, J=19.4 Hz),−21.77 (t, J=19.4 Hz);

ToF-MS (ESI): For the molecular ion C₂₁H₂₅N₆O₁₂P₃Na [M−2H+Na]⁻, thecalculated mass was 669.0641, and the observed mass was 669.0960.

6-FAM labeledN⁶-{1-[4-(3-Amino-1-propynyl)phenyl]ethyl}-2′-deoxyadenosine-5′-triphosphate(WW2p093)

A solution of 6-FAM-SE (3.5 mg, 7.4 mmol) in anhydrous DMSO (70 4) wasadded to a solution of triphosphate dA.n14 (4.1 mmol) in Na₂CO₃/NaHCO₃buffer (0.1 M, pH 9.2; 600 μL) and incubated at room temperature for onehour. The reaction was purified by reverse-phase HPLC using a PerkinElmer OD-300 C₁₈ column (4.6×250 mm) to yield the 6-FAM labeledtriphosphate WW2p093. Mobile phase: A, 100 mM triethylammonium acetate(TEAA) in water (pH 7.0); B, 100 mM TEAA in water/CH₃CN (30:70). HPLCpurification was achieved using a linear gradient of 5-20% B for 20minutes and then 20-90% B for 20 minutes. The concentration of WW2p093was estimated by adsorption spectroscopy using the extinctioncoefficient of the 6-FAM dye (i.e., 68,000 at 494 nm).

All patents and patent publications referred to herein are herebyincorporated by reference. Certain modifications and improvements willoccur to those skilled in the art upon a reading of the foregoingdescription. It should be understood that all such modifications andimprovements have been deleted herein for the sake of conciseness andreadability but are properly within the scope of the following claims.

1. A compound according to the following formula or salt thereof:

wherein R₁ is H, monophosphate, diphosphate, triphosphate, oralpha-thio-triphosphate, R₂ is H or OH, base is cytosine, uracil,thymine, adenine, guanine, or a naturally occurring derivative thereof,non-cleavable terminating moiety is a group imparting polymerasetermination properties to the compound, linker is a bifunctional group,and dye is a fluorophore.
 2. The compound of claim 1, wherein thenon-cleavable terminating moiety is attached to the base through alinkage selected from the group consisting of benzyl amine, benzylether, carbamate, carbonate, 2-(o-nitrophenyl)ethyl carbamate, and2-(o-nitrophenyl)ethyl carbonate.
 3. A compound selected from the groupof formulas or salts thereof consisting of:

wherein R₁═H, monophosphate, diphosphate, triphosphate, oralpha-thio-triphosphate, R₂═H or OH, R₃ and R₄ are each independentlyselected from the group consisting of H, a C₁-C₁₂ straight chain orbranched alkyl, a C₂-C₁₂ straight chain or branched alkenyl or polyenyl,a C₂-C₁₂ straight chain or branched alkynyl or polyalkynyl, and anaromatic group, R₅, R₆, and R₇, are each independently selected from thegroup H, OCH₃, NO₂, CN, a halide, a C₁-C₁₂ straight chain or branchedalkyl, a C₂-C₁₂ straight chain or branched alkenyl or polyenyl, a C₂-C₁₂straight chain or branched alkynyl or polyalkynyl, and an aromaticgroup, and/or with the proviso that one of R₅, R₆ and R₇ is a linkergroup of the general structure —C≡CCH₂NH₂,

wherein X═CH₂, CH═CH, O, S, or NH, Y═CH₂, O, or NH, n=an integer from0-12; m=an integer from 0-12, Dye=a fluorophore, and R₈ and R₉ are asdefined above for R₅, R₆, and R₇, with the proviso that R₈ and R₉ arenot NO₂.
 4. The compound of claim 3, wherein R₃ and R₄ are eachindependently selected from the group consisting of H, —CH₃, —CH₂CH₃,—CH₂CH₂CH₃, isopropyl, tert-butyl, and phenyl.
 5. The compound of claim3, wherein R₃ and R₄ are each independently selected from the groupconsisting of H, alkyl and aromatic groups optionally containing atleast one heteroatom in the alkyl or aromatic groups, and furtherwherein the aromatic group may optionally be an aryl or polycyclicgroup.
 6. The compound of claim 3, wherein R₅, R₇, R₈, and R₉ are eachH. 7-8. (canceled)
 9. A method of determining the sequence of a targetnucleic acid comprising (i) adding a target nucleic acid to a Sanger orSanger-type sequencing apparatus, (ii) adding one or more compoundsaccording to claim 1 to the sequencing apparatus, with the proviso thatwhere more than one type of base is present, each base is attached to adifferent fluorophore; (iii) adding a complementary primer and apolymerase enzyme, (iv) performing a polymerase reaction to incorporateat least one of the compounds of step (ii) into a growing nucleic acidstrand, and (v) analyzing the result of the Sanger sequencing reactionwith fluorescence sequencing instrumentation or by pulsed multilineexcitation fluorescence, wherein steps (i)-(iii) can be performed in anyorder.
 10. The method according to claim 9, wherein incorporation of atleast one compound according to step (iv) is followed by termination ofstrand growth at an efficiency of from about 90% to about 100%.
 11. Themethod according to claim 9, wherein the incorporation of at least onecompound according to step (iv) occurs at about 70% to about 100% of theefficiency of incorporation of a native substrate with the same base inthe polymerase reaction.
 12. The method according to claim 11, whereinthe incorporation efficiency occurs at about 85% to about 100%.
 13. Themethod according to claim 9, wherein the polymerase is selected from thegroup consisting of reverse transcriptase, terminal transferase, and DNApolymerase. 14-15. (canceled)
 16. A method of incorporating anon-naturally occurring component into a nucleic acid comprising: (i)adding a target nucleic acid to a sequencing apparatus; (ii) adding oneor more compounds according to claim 1 to the sequencing apparatus, withthe proviso that where more than one type of base is present, each baseis attached to a different fluorophore; (iii) adding a polymeraseenzyme; and (iv) performing a polymerase reaction to incorporate atleast one of the compounds of step (ii) into a growing nucleic acidstrand, wherein steps (i)-(iii) can be performed in any order.
 17. Themethod according to claim 16, further comprising (v) analyzing theresult of the polymerase chain reaction for incorporation of at leastone compound from step (ii).
 18. The method according to claim 16,wherein incorporation of at least one compound according to step (iv) isfollowed by termination of strand growth at an efficiency of from about90% to about 100%.
 19. The method according to claim 16, wherein theincorporation of at least one compound according to step (iv) occurs atabout 70% to about 100% of the efficiency of incorporation of nativesubstrate with the same base in the polymerase reaction.
 20. A method ofterminating nucleic acid synthesis comprising the step of placing acompound according to claim 1 in the environment of a polymerase andallowing incorporation of the compound into a nucleic acid.
 21. Themethod according to claim 20 wherein the efficiency of termination uponincorporation of the compound ranges from about 90% to about 100%. 22.The method according to claim 20 wherein the efficiency of incorporationof the compound ranges from about 70% to about 100% compared to theefficiency of incorporation of a naturally-occurring nucleotide ornucleoside with the same base.
 23. A method of performing Sanger orSanger-type sequencing comprising addition of a compound according toclaim 1 to a Sanger or Sanger-type sequencing method.
 24. A method ofperforming mini-sequencing or minisequencing-type sequencing comprisingaddition of a compound according to claim 1 to a mini-sequencing orminisequencing-type sequencing method.